Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V017894 | pLV3-U6-MCS-sgRNA-EF1a-EGFP-Puro | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
- Vector Name:
- pLV3-U6-MCS-sgRNA-EF1a-EGFP-Puro
- Antibiotic Resistance:
- Ampicillin
- Length:
- 10957 bp
- Type:
- Gene-editing vector
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Puro
- Promoter:
- U6
- 5' Primer:
- U6-F
- Growth Strain(s):
- Top10
- Growth Temperature:
- 37℃
pLV3-U6-MCS-sgRNA-EF1a-EGFP-Puro vector Map
Plasmid Protocol
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5. Store the plasmid at -20 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
pLV3-U6-MCS-sgRNA-EF1a-EGFP-Puro vector Sequence
LOCUS . 10957 bp DNA circular UNK 01-JAN-1980
DEFINITION synthetic circular DNA.
ACCESSION
VERSION
KEYWORDS .
SOURCE .
ORGANISM .
.
FEATURES Location/Qualifiers
promoter 87..1265
/label="EF-1α promoter"
/note="strong constitutive promoter for human elongation
factor EF-1α"
CDS 1941..2657
/label="EGFP"
/note="the original enhanced GFP (Yang et al., 1996)"
misc_feature 2667..3255
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 3327..3560
/label="3' LTR (ΔU3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 3638..3772
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 3799..3934
/label="SV40 ori"
/note="SV40 origin of replication"
promoter complement(3955..3973)
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
rep_origin 4141..4596
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 4622..4726
/label="AmpR promoter"
CDS 4727..5584
/label="AmpR"
/note="β-lactamase"
rep_origin 5758..6346
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 6634..6655
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6670..6700
/label="lac promoter"
/note="promoter for the E. coli lac operon"
promoter 6769..6787
/label="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 6815..7041
/label="RSV promoter"
/note="Rous sarcoma virus enhancer/promoter"
LTR 7042..7222
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 7269..7394
/label="HIV-1 Ψ"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 7887..8120
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 8305..8349
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
promoter 8527..8767
/label="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 10657..10732
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
misc_feature 10788..10905
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"